CN102743770B

CN102743770B - A kind of targeting molecule image probe and living body molecule imaging method

Info

Publication number: CN102743770B
Application number: CN201210200690.6A
Authority: CN
Inventors: 申宝忠; 程震; 卜丽红
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-06-18
Filing date: 2012-06-18
Publication date: 2017-07-21
Anticipated expiration: 2032-06-18
Also published as: CN102743770A; US9764048B2; US20150202335A1; WO2013189113A1

Abstract

The present invention relates to medical technical field, more particularly to a kind of living body molecule imaging method and targeting molecule image probe.Living body molecule imaging method of the present invention reflects disease of cardiovascular system in the form of images by introducing the targeting molecule image probe for being available for iconography equipment to detect（Especially arrhythmia cordis）With the inserted by connexin 43 of the disease such as tumour（Cx43）Biochemical change feature, testing result is accurate, can intuitively show Cx43 distribution and quantity, may be directly applied to human body.Cx43 targeting molecules image probe disclosed by the invention, targets affine component by signal component, Cx43 and the part of connector three is constituted, with good Pharmacokinetic Characteristics and biodistribution feature.

Description

A kind of targeting molecule image probe and living body molecule imaging method

Technical field

The present invention relates to medical technical field, more particularly to a kind of Cx43 targeting molecules image probe and living body molecule into Image space method.

Background technology

Gap Junction Remodelling（gap junction remodeling）It is to participate in arrhythmia cordis, tumour, atherosclerosis One of common pathologic basis occur etc. disease, developed, and inserted by connexin 43（Connexin43, Cx43）It is then composition seam The fundamental structural unit of gap connection, is the primary structure basis of gap connection between composition cardiac muscle cell.Cx43 is that molecular weight is A kind of 43Kd connection albumen, its main function is to mediate the Direct Communication between flanking cell.In heart, its main function is Form iuntercellular quickly electricity impulsion, it is ensured that heart entirety electrical activity synchronism and harmony, maintain the electrical activity of cardiac muscle cell And the synchronism of Mechanical Contraction and diastolic function.Therefore, Cx43 is sent out in terms of quantity, distribution, function and phosphorylation state It is raw to change（That is Gap Junction Remodelling）When will necessarily cause electrical coupling obstacle between cardiac muscle cell.It is mainly shown as iuntercellular electrical activity Synchronism, coordinate sexually revise, conducting speed slow down and it is anisotropic change etc..A variety of acquired angiocarpy of adult Disease, such as myocardial ischemia, arrhythmia cordis, heart failure, hypertension, atherosclerosis occur in that different degrees of Cx43 seams Gap connection reconstruct.Cx43 Gap Junction Remodellings are various arrhythmia cordis, the important feature that especially reciprocal tachycardia is produced Basis, is also the main reason of various heart disease deaths and sudden death.

Meanwhile, Cx43 participates in various kinds of cell life cycle from the links for growing into death, direct mediate cell growth Transmission of the adjustment signal between flanking cell, regulating cell growth, differentiation and apoptosis.Cx43 exception will result directly in cell Adjusting and controlling growth is abnormal, and contact inhibition, terminal differentiation and apoptosis capacity disappear, and shows as extension or immortality cell life Cycle, so as to cause tumour.

Therefore, Cx43 is used as antiarrhythmic therapy and the common molecular target spot of antineoplaston, it has also become domestic unfaithful intention Vascular diseases and the new study hotspot in tumor research field.A variety of study in vitro are also developed accordingly, are used for Cx43 quantity and function（Including phosphorylation state）Analysis.These research methods include：Using PCR or RT-PCR in mRNA water Flat detection Cx43 expression；Semidefinite quantity research is carried out to Cx43 albumen using Western Blot；It is glimmering by immuning tissue Light staining technique, observation Cx43 subcellsular level positioning and Gap Junction Remodelling etc.；By introducing the different phosphorylation positions of Cx43 The specific antibody of point, utilizes Matrix Assisted Laser Desorption/MALDI-MS, immobilized metal affinity chromatography, liquid phase tandem mass spectrum point Analyse the phosphorylation state of Cx43 different phosphorylation sites；Tested using fluorescent dye intercellular trafficking, by microinjection side Small molecule fluorescent dyestuff is incorporated into cytoplasm by method, and with reference to fluorescence microscopy, the thin of Cx43 mediations is evaluated in cellular level Intercellular Direct Communication function etc..But, these external detection methods are relied primarily on to substantial amounts of, cellular level or in vitro tissue The analysis of experimental result.

Though external detection method can determine whether, analyze the Cx43 expressions and functional status of cell or tissue, from technology Analyzed in aspect, all there is certain limitation：

1. need to obtain sample by cell culture, biopsy or postmortem, it is impossible to directly apply to human body；

2. Vitro Experimental Results may not be inconsistent with the truth under condition of living organism：Experiment in vitro is by experiment condition, experiment Equipment, experimental method influence it is larger, in the processing procedure of sample, some important compositions may be lost, error is larger, So that experimental result is not inconsistent with the truth under condition of living organism；

3. experiment in vitro is unfavorable for dynamic studies：Experiment in vitro needs to put to death experimental animal to obtain at different time points Tissue or repeatedly biopsy extraction, can only observe a certain stage of disease, it is impossible to realize that really dynamic is ground in same animal body Study carefully, be difficult to obtain accurate conclusion in the disease overall process of such complicated, progress；

4. complex operation, much time wealth, randomness is very big.Therefore, Cx43 live body visual research may be to solve this A little problems are there is provided a kind of new thinking, and the appearance of molecular imaging technology makes possibility.

Molecular imaging refers to that under condition of living organism image application method is to the cell and molecular water in human or animal's body Flat biological process is imaged, qualitative and quantitative study a subject.It is used using application molecular probe as distinguishing feature A variety of imaging means, are imaged to internal particular target point.Imaging means include：Radionuclide imaging（radionuclide imaging）, magnetic resonance imaging（Magnetic resonance imaging, MRI）, magnetic resonance spectrum imaging（MR Spectroscopy, MRS）, optical imagery（Optical imaging, OI）, ultrasonic imaging（Ultrasound imaging, US）And multi-pattern Fusion imaging（integration of multi-mode imaging）Deng.It is raw by these imaging techniques The life some specific physiology of internal system or pathologic process, such as gene expression, the interphase interaction of protein, signal transduction, The metabolism of cell and Cellular tracking etc. can be changed into intuitively image and display.

The Cx43 visualization schemes of the progress such as V Baklaushev, have been synthesized with E2 sections outside Cx43 albuminous cells of specificity Antibody is targets affine component, with radio isotope¹²⁵I and fluorescent dye Alexa660 is the probe of signal component, utilizes γ Ray counter and fluorescence microscope carry out the abnormal expression situation of Cx43 in detection glioma, but still without breaking away from external inspection The limitation of checking method：

①¹²⁵I is not a kind of nucleic of suitable animal scanning, can only be detected using gamma radiation counter, and is checked As a result it is not directly perceived enough.So the research is in intravenous injection probe¹²⁵After I-MAbE2Cx43, animal is put to death, tissue is obtained, using γ Ray counter has carried out radioassay；

2. Alexa660 is not the fluorescent dye suitable for living imaging yet, is only used for immuning tissue's staining analysis, Therefore the research puts to death animal after intravenous injection probe Alexa660-MAbE2Cx43, obtains tissue, frozen section is made, Tissue is analyzed using fluorescence microscope；

3. using MAbE2Cx43 antibody as affine component, antibody is expensive, and may cause immunogenic response So as to trigger side effect.Importantly, because antibody molecule amount is larger, non-specific adsorption can be higher, after being injected into vivo It is difficult to obtain preferable Pharmacokinetic Characteristics and biodistribution feature, causes testing result inaccurate, application is not square Just.

The method for needing In vivo detection and quantitative Cx43 badly at present.

The content of the invention

Present invention mainly solves technical problem be exactly Cx43 In vivo detection and quantitative problem.It is imaged by living body molecule Technology carried out In vivo detection to Cx43 and quantitative, and Cx43 expression quantity, distribution, function and disease development can be determined in live body During Cx43 dynamic change situation, specify Cx43 targeted therapies intervention Best Times, dosage, and to " rebuild Cx43 " control The curative effect for the treatment of carries out accurate objective appraisal, so as to realize that the prognosis of the diseases such as Rapid exhumation arrhythmia cordis and malignant tumour is commented Estimate, Cx43 targeted therapy curative effect monitorings.

On the one hand, the present invention provides a kind of method of living body molecule imaging, and methods described includes：

Cx43 targeting molecule probes are provided, the targeting molecule probe by signal component, target affine component and The part of connector three constitute, the signal group be divided into be available for iconography equipment detect part, the affine component of targeting be with The part of Cx43 specific bindings, the connector connects signal component and the affine component of targeting；

Implement optical imagery, positron emission to patient position to be measured with described Cx43 targeting molecules probe to break Layer imaging, single photon emission tomographic imaging, magnetic resonance imaging, photoacoustic imaging, ultrasonic imaging or other live body iconographies are fused into As technology preferred PET/CT, PET/MRI.

Preferably, the affine component of targeting and Cx43 carboxyl terminal are specifically bound.It is highly preferred that the targeting Affine component is selected from：

Ith, Cx43SP1, Gly-Ala-Pro-Gly-4Hyp-Pro-Tyr

IIth, Cx43SP2, Gly-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly

IIIth, Cx43SP3, its structural formula is as follows：

IVth, Cx43SP4, including 5 kinds have RXP-X structures analog, it is specific as follows：

RXP-A, its amino acid sequence is as shown in SEQ ID No.1；

RXP-B, its amino acid sequence is as shown in SEQ ID No.2；

RXP-C, its amino acid sequence is as shown in SEQ ID No.3；

RXP-D, its amino acid sequence is as shown in SEQ ID No.4；

RXP-E, its amino acid sequence is as shown in SEQ ID No.5.

Preferably, the signal group is selected from radio isotope, fluorescent dye, quantum dot, paramagnetism material, magnetic Property nano-particle, superparamagnetic material, ultrasonic microbubble, the one or more of optoacoustic nano particle.

Preferably, the connector be selected from DTPA, DOTA, DOTAGA, NOTA, NODAGA, TETA, CB-TE2A, Sar, The chelating agents such as NODA or other chemical methodes that directly can be joined directly together signal component and the affine component of Cx43 targetings.

On the other hand, the present invention provides a kind of targeting molecule probe, by signal component, targets affine component and will letter Number component and the part of connector three composition that affine component is connected is targetted, the signal group, which is divided into, is available for iconography equipment to examine The part of survey, the affine component of targeting is the part specifically bound with Cx43, and the connector is by signal component and targeting Affine component is connected.

Preferably, the affine component of targeting of the targeting molecule probe and Cx43 carboxyl terminal are specifically bound.

It is highly preferred that the affine component of targeting is selected from：

Ith, Cx43SP1, Gly-Ala-Pro-Gly-4Hyp-Pro-Tyr

IIth, Cx43SP2, Gly-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly

IIIth, Cx43SP3, its structure is as follows：

IVth, Cx43SP4, including 5 kinds of analogs with RXP-X structures, sequence is as follows：

RXP-X	Sequence
		RXP-A	DVPGRDPGYIKGGGSAHARVPFF SHSLNRNRKP SLYQ
RXP-B	EIQPRSPLMFSGGGSAHARVPFFSHSAKEARWPRAHR
		RXP-C	GIAAREPNSHDGGGSAHARVPFFSHSRDLWRKPAKSL
RXP-D	WEEPRRPFTMSGGGSAETHARVPFYSHSPMRHRLPGVHL
		RXP-E	SDDLRSPQLHNGGGSAVPFYSHSHMVRRKPRNPR

Preferably, the connector be selected from DTPA, DOTA, DOTAGA, NOTA, NODAGA, TETA, CB-TE2A, Sar, The chelating agents such as NODA, or be directly connected to signal component and the affine components of Cx43 using other direct chemical reactions.

Molecular probe（molecular probe）：Being can be with a certain specific biological molecules（Such as protein, DNA, RNA） Or eucaryotic cell structure targeting specific combination and be available in vivo or（With）The labeled compound molecule of external iconography spike, These labeled compound molecules can in live body or（With）Reflect in vitro its target biomolecule amount and（Or）Function.Can be simple It is interpreted as molecular imaging diagnostics classes medicine.

Molecular imaging probe must possess following 2 key characters：Pair 1. there is height with the closely related target molecule of disease Spend affinity and targeting specific；2. it is available for iconography equipment in vitro to carry out spike.Probe is mainly used in right in vivo Bioprocess is imaged, quantitative and measuring study.Basic structure is generally divided into three parts：Signal component（signaling component）, affine component（affinity component）And connector（linker）.Signal component refers to that shadow can be produced As learning signal and the contrast agent that can be detected by high-precision imaging technique or label part（Such as radionuclide, fluorescein, suitable Magnetic atom and ultrasonic microbubble etc.）；Affine component is targeted molecular, is the part with imaging target spot specific binding（Such as part Or antibody etc.）.Directly signal component and affine component can be connected by radio chemistry or biomolecule click chemistry technology Pick up and, can also pass through linker, i.e. introducing crosslinked reagent or derivatization reagent（crosslinking or derivatizing reagents）The two is connected.

The present invention provides a kind of molecular targeted specific probes of Cx43, such probe by radio isotope, fluorescent dye, The Imaging Methods such as quantum dot, nano-particle, magnetic material, ultrasonic microbubble, photoacoustic imaging material and multi-modal imaging are marked Cx43 targeting specific Binding peptides are formed.Such probe is introduced, using positron emission tomography（PET）, single photon hair Penetrate tomography（SPECT）, the image such as optical imagery, ultrasonic imaging, magnetic resonance imaging, photoacoustic imaging and multi-modal imaging learns to do Section can be detected in vivo under normal physiological and pathologic condition, the Cx43 of area-of-interest quantity, distribution and function, and its The variation characteristic of the different phase developed in disease.

Have confirmed that the probe in vivo has good stability through research, Cx43 targeted moleculars imaging specificity is high, accurately Property it is high, the contrast of image is good, it is adaptable to disease of cardiovascular system（Especially arrhythmia cordis）, tumour diagnosis, Cx43 targeting Curative effect monitoring is treated, Best Times, the dosage of targeted therapy intervention is specified, and to " the curative effect progress for rebuilding Cx43 " treatments is accurate Objective appraisal.Meanwhile, the also basic research for tumour and disease of cardiovascular system provides effective method, for research Cx43 The correlation between generation, Characteristics of Development with disease provides new means.

The present invention also provides a kind of image forming composition, includes above-mentioned targeting molecule probe.

The present invention also provides the targeting molecule probe in the reagent for preparing diagnosis Cx43 abnormal expression relevant diseases Purposes.

Preferably, the Cx43 abnormal expressions relevant disease is using Cx43 expression or dysfunction as the disease of characterization of molecules Disease, mainly includes arrhythmia cordis or tumour.

The present invention sets up noninvasive, intuitively Cx43 living body molecules imaging method, and synthesis is available for the spy that iconography equipment is detected Pin, intuitively shows Cx43 distribution and quantity, its function of preliminary assessment, and the technology, which has, to be set up safe, noninvasive, live body, moves State, feature that is directly perceived, accurate, may be directly applied to human body.

The class Cx43 targeting imaging diagnosis class medicines that the present invention is developed（Molecular probe）, moved with good medicine generation Mechanical characteristics and biodistribution feature, testing result are more accurate, using more facilitating, in the form of images the reflection heart directly perceived Restrain the biochemical characteristics of the diseases such as not normal and tumour.

The targeting specific probe of the present invention has the following advantages that：

1) has Cx43 targeting specifics, it is ensured that the accuracy of Cx43 molecular imagings targeting detection

Non-specific probe used in general iconography and Nuclear medicine is unable to the life in specific recognition and combination Thing molecule, therefore the information that disease molecules change downstream can only be provided（Dissection, pathology and physiology change）, or disease is whole The change of volume morphing and function information, such as CBF, the change of blood perfusion.If thinking accurately to detect in disease progression Key point thing molecule, then require probe can in specific recognition and combination molecular marker there is provided disease molecular level Information, deepens the understanding to disease organism process.In addition, using targeting specific probe, probe and other can be efficiently reduced The non-specific binding of non-imaged target spot, more contributes to that imaging target spot is carried out accurately to quantify.

2) .Cx43 live body visualization, it is ensured that safe, the noninvasive and intuitive of Cx43 molecular imagings detection

Realize the live body visualization of key molecule mark in disease progression, it is desirable to which probe has transmitting iconography letter Number, the performance for being available for noninvasive iconography equipment to detect in vitro intuitively shows the distribution of molecular marker by image Situation.Because the characteristics of imageological examination has safe, noninvasive, directly perceived.

3) high-affinity of molecular probes and target spot Cx43

Cx43 In vivo detections are realized, the premise for obtaining preferable molecular imaging image is height of the molecular probe in imaging target spot Concentration is assembled, that is, requires that probe reaches behind target area early stage just and targeted integration, the time of dissociation is relatively later, it is ensured that in several blood After liquid cycle period, probe reaches preferable coherent condition in target spot.

4) molecular probes detect Cx43 hypersensitivity

In the situation of change for early detecting molecular marker of disease early stage, or Results, probe is usually required that Very small amount of biomarker is can detect, i.e., with high susceptibility.In addition, preferable molecule different from medicine Probe must assure that its biological impact produced or pharmacotoxicological effect are as far as possible low, it is therefore desirable to which the sensitiveness of probe is sufficiently high, Only need to a small amount of probe and be achieved with preferable image, the quantity for introducing intracorporeal probe is reduced as far as possible, reduction probe triggers Pharmacotoxicological effect.

5) high-contrast of .Cx43 living body molecules imaging

The signal intensity of the image request lesion region of high-contrast is sufficiently high, i.e. target/background ratio and signal/noise ratio Value is very high.This requires probe to have preferable biodistribution feature, i.e. probe big in the dense poly- amount in target area, residence time It is long, and uptake ratio is low in normal structure organ, removing speed is fast.

6) the live body stability of molecular probes

Although the introduction volume of molecular probe seldom may maintain the stability and integrality of molecular probe in vivo A problem is still, because there are many enzymes in blood plasma or in target tissue, probe can be degraded.Picture quality and quantitatively grind The accuracy studied carefully depends on the stability of probe in vivo.

7) immunogenicity and toxicity of molecular probes are low

Applied to human body molecular probe must safely, without immunogenicity and toxicity.The pharmacotoxicological effect of generation is tried one's best It is low.

8) production process of molecular probes is simple and easy to do, and cost is low

The preparation process of molecular probe is convenient and easy, with low cost, helps to be widely applied in clinic.If produced Journey is complicated, and cost height will certainly then influence the clinical conversion of molecular probe.

Brief description of the drawings

Fig. 1 is the structural representation of Cx43 targeted molecular image probes；

Fig. 2 is the fluorescent microscopy images of probe in vitro with HeLa-Cx43 cell binding experiments：

a：After Cy5.5-Cx43SP and HeLa-Cx43 cells are incubated altogether, the substantial amounts of probe of HeLa-Cx43 cellular uptakes Cy5.5-Cx43SP（Green）；

b：After Cy5.5-Cx43SP is incubated altogether with HeLa cells, Cy5.5-Cx43SP is not by control group HeLa cellular uptakes；

c：Introduce excessive un-marked Cx43SP to block in advance, then Cy5.5-Cx43SP and HeLa-Cx43 cells are total to After incubation, HeLa-Cx43 cellular uptakes Cy5.5-Cx43SP amount is significantly reduced, and is not almost absorbed；

d：Introduce excessive un-marked Cx43SP to block, after Cy5.5-Cx43SP is incubated altogether with HeLa cells, HeLa is thin Born of the same parents do not absorb Cy5.5-Cx43SP still.Confirm that probe has good targeting specific.

Fig. 3 is tissue freezing section's fluorescent microscopy images using muscle as control:

a：Shown under 1h after tail vein injection Cy5.5-Cx43SP, muscle frozen section fluorescence microscope, Cy5.5- Cx43SP is in musculature without aggregation;

b：Muscle frozen section bright field pictures, show musculature morphology;

c：Muscle frozen section and fluorescence microscope figure below fusion picture.

Fig. 4 be Active MnO2 probe after, cardiac muscular tissue's frozen section fluorescent microscopy images：

a：Picture, Cy5.5- under 1h after tail vein injection Cy5.5-Cx43SP, myocardium frozen section fluorescence microscope Cx43SP largely assembles in cardiac muscular tissue;

b：1h after tail vein injection Cy5.5-Cx43SP, myocardium frozen section bright field pictures show cardiac muscular tissue Form；

c：1h after tail vein injection Cy5.5-Cx43SP, myocardium frozen section and fluorescence microscope figure below fusion picture is shown Cy5.5-Cx43SP largely assembles in cardiac muscular tissue, and is distributed mainly on position where the connection of gap between cardiac muscle cell.

Fig. 5 is⁶⁴Cu-NODA-Cx43SP1 is combined and blocking experiment with Hela-Cx43 cells

Fig. 6 is in vitro Main Tissues organ near-infrared fluorescence imaging：After mouse tail vein injection probe Cy5.5-Cx43SP 1h, probe is significantly built up in heart, stomach, liver, gall-bladder and enteron aisle, most of to be drained through liver sausage road.

Fig. 7 is⁶⁴The PET imagings of Cu-NOTA-Cx43SP1 different time points in normal rat, display probe is poly- in heart Collection is obvious.⁶⁴The microcurie of Cu-NOTA-Cx43SP1 injection volumes about 200.

Fig. 8 is⁶⁴Cu-NOTA-Cx43SP1 PET of 1 hour in normal mouse are imaged, and display probe assembles bright in heart It is aobvious.⁶⁴The microcurie of Cu-NOTA-Cx43SP1 injection volumes about 50.

Fig. 9：Intravital mouse tumour near infrared imaging is tested：1h after mouse tail vein injection probe Cy5.5-Cx43SP1, The HeLa-Cx43 tumor locus for being overexpressed Cx43 is largely assembled, and in right side control group HeLa tumor locus without dense poly-.

Figure 10：Internal mouse tumor PET imaging experiments：Mouse tail vein injection probe⁶⁴1h after Cu-NODA-Cx43SP1, Probe is largely assembled in the HeLa-Cx43 tumor locus for being overexpressed Cx43（Left side）.

Embodiment

The invention discloses a kind of Cx43 targeting molecules image probe and living body molecule imaging method, people in the art Member can use for reference present disclosure, be suitably modified technological parameter realization.In particular, all similar replacements and change Apparent to those skilled in the art, they are considered as being included in the present invention.The method of the present invention and application Be described by preferred embodiment, related personnel substantially can not depart from present invention, in spirit and scope it is right Method described herein and application are modified or suitably change is with combining, to realize and apply the technology of the present invention.

In order that those skilled in the art more fully understands technical scheme, with reference to specific embodiment pair The present invention is described in further detail.

Embodiment 1：Cx43 targets affine component -- Cx43 targeting binding peptides（Cx43SP）

4 class polypeptide below（Cx43 targeting binding peptides）Any kind can be tied with Cx43 carboxyl terminal specificity Close, therefore can be used to synthesize Cx43 targeted molecular probes.

1）, Cx43SP1 structure：

Gly-Ala-Pro-Gly-4Hyp-Pro-Tyr, also known as：AAP10, molecular formula：C₂₆H₃₇N₇O₈, molecular weight： 575.6, its structure is as follows：

Cx43SP1 is the most important affine component of targeting in molecular probe of the present invention, be a kind can be specific in vivo Recognize Cx43, and the polypeptide of 6 amino acid composition in combination.Cx43SP1 is Aonuma S in 1980 etc. in the atrium of ox A series of anti-arrhythmia polypeptides by acting on Cx43 and working extracted in tissue, therefore be once named as antiarrhythmic peptide10(AAP10).But it is due to AAP10 structural instabilities in blood plasma, fails to have further Clinical practice.Invention person has carried out chemical modification to AAP10 structures, has synthesized the spy for Cx43 living imagings Pin, and demonstrate the probe there is preferable live body stability.Because in the present invention, the chemical constitution is not re-used as the anti-rhythm of the heart Arrhythmic agents, but as the affine component of targeting of Cx43 targeted molecular image probes, therefore it is named as Cx43 specific bindings Peptide 1 (Cx43 Specific Peptide 1).Cx43SP1 horseshoe-shaped structure domain can be with Cx43 carboxyl terminal acceptor knot Structure domain is specifically bound.

2）, Cx43SP2 structure：

Gly-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly, its structure is as follows：

Cx43SP2 is Cx43SP1 analog, and the part L-type amino acid in Cx43SP is instead of with D type amino acid, from And stability is added, have been introduced into clinical experimental stage currently as antiarrhythmic drug.Cx43SP2 trade name： Rotigaptide, also known as：CHEMBL450656, GAP-486, ZP123.Molecular formula：C₂₈H₃₉N₇O₉, molecular weight：617.65076.

3）Cx43SP3 structure：

Cx43SP3 is Cx43SP2 functional analogue, also known as GAP 134, and molecular weight is 291.3, the following institute of its structure Show：

4）Cx43SP4 is 5 kinds of amino acid sequences comprising RXP-E characteristic structurals, as shown in table 1：

The Cx43SP4 of table 1 amino acid sequence

RXP-X	Sequence
		RXP-A	DVPGRDPGYIKGGGSAHARVPFFSHSLNRNRKPSLYQ
RXP-B	EIQPRSPLMFSGGGSAHARVPFFSHSAKEARWPRAHR
		RXP-C	GIAAREPNSHDGGGSAHARVPFFSHSRDLWRKPAKSL
RXP-D	WEEPRRPFTMSGGGSAETHARVPFYSHSPMRHRLPGVHL
		RXP-E	SDDLRSPQLHNGGGSAVPFYSHSHMVRRKPRNPR

The class that Cx43SP3, which is Mario Delmar et al. 2006, to be screened by display technique of bacteriophage can be with Cx43 carboxyl terminal（Amino acid 255-382）The polypeptide of specific binding, by 34 Amino acid profiles.It is basic to be with reference to block RXP-X, therefore be called and do the serial polypeptides of RXP, it is envisaged for antiarrhythmic therapy.Wherein RXP-E has more compared with other polypeptides should Use prospect.

Embodiment 2：Prepare Cx43 targeting molecule probes

The general structure of Cx43 targeting molecules probe of the present invention is as shown in Figure 1.

（1）Imaging target spot is inserted by connexin 43 (Connexin 43, Cx43), and Cx43 carboxyl terminal (C-terminal) has One gate nutty structure, function equivalent to specific receptor domain, be Cx43SP mainly in combination with target spot, be this hair The target spot of bright Cx43 targeted imagings.

（2）Signal component：It is the part that probe is available for iconography equipment to detect, predominantly radio isotope in this patent （PET and SPECT imagings）, fluorescent dye and quantum dot（Optical imagery）, paramagnetic material and superparamagnetic material and magnetic receives Rice corpuscles（Magnetic resonance imaging）, ultrasonic microbubble（Ultrasonic imaging）, various optoacoustic nano particles（Photoacoustic imaging）And any of the above group Divide the iconography material for the multi-modal imaging technology for detection being combined.

（3）Target affine component：It is probe and the part of the molecular target specific binding of imaging, combination therebetween With high degree of specificity and high-affinity, equivalent to the relation of " key and lock ".It is mainly concerned with the present invention in embodiment 1 Polypeptide and small molecule structure.

（4）Connector：The part that signal component and the affine component of targeting are connected.Also connector can not be introduced, and It is directly to be directly connected to signal component and the affine components of Cx43 using chemical method.

Embodiment 3：The preparation of the Cx43 targeted molecular probes of labelled with radioisotope

4 class polypeptides can be specifically bound with Cx43 carboxyl terminal in embodiment 1, therefore be can use and be prepared into mark Cx43 targeted molecular probes.In following building-up process citing, representative polypeptide is used as with Cx43SP1.

Signal group is divided into positron emitting radio isotope or Single Photon Emission nucleic, available for clinical and small Animal positron emission tomography (PET) or single photon emission computerized tomgraphy（SPECT）.The appropriate connector of selection, passes through Radio chemistry method can link together radio isotope and targeted polypeptide.The connector of use, which is called, does difunctional Chelating agent.Bifunctional chelating agent both had the function block group combined with radioactive metal, also there is the base combined with Cx43SP Group, the two is connected, as Cx43 targeting molecule probes.

1）Divalence（M²⁺）Or trivalent metal ion（M³⁺）Mark Cx43SP

It can select different connectors according to radionuclide different qualities, refer to table 2.

Thus table 2 often synthesizes with radio isotope, bifunctional chelating agent and Cx43 targeting probes

Note：R is Cx43SP.

DTPA：Diethylene triamine pentaacetic acid diethylenetriamine pentaacetic acids

DOTA：1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1,4,7, 10- tetraazacyclododecanand -1,4,7,10- tetraacethyls

DOTAGA：

1-(1-carboxy-3-carboxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7, 10-triacetic acid

1- (1- carboxyl -3- carboxylics propyl group)-Cyclen -1,4,7,10- tetraacethyls

NOTA：1,4,7-triazacyclononane-1,4,7-triacetic acid 1,4,7- sodium azide -1,4, 7- triacetic acids

NODAGA：NOTA carries out functionalized modification by glutaric acid arm

2-(p-SCN-Bz)-NOTA：NOTA carries out functionalized modification by benzyl isothiocyanic acid

TETA：1,4,8,11-tetraazacyclotetradecane-1,4,8,11,tetraacetic acid1,4, 8,11- macrocyclic tetraamines tetraacethyl -1,4,7,10- tetraacethyls

CB-TE2A：Attachment of two carboxymethyl pendant arms to cross-bridged (CB)-cyclam leads to CB-TE2A

Sar：sarcophagine(3,6,10,13,16,19-hexa azabicyclo[6.6.6]icosane)

Cobalt ions（3,6,10,13,16,19- hexabromos azabicyclo [6.6.6] icosane）

SarAr and AmBa Sar are Sar carboxylic acids and aminoderivative

NODA：1,4,7-triazacyclononane-1,4-diacetate 1,4,7- sodium azide -1,4- diacetic acids Sodium

Due to a variety of radio isotopes and bifunctional chelating agent referred to above, herein with⁶⁴Exemplified by Cu marks Cx43SP, Illustrate radioisotopic divalence（M²⁺）Or trivalent metal ion（M³⁺）The method for marking Cx43SP.

NODA modifies Cx43SP:NODA is excessive, plus appropriate DMF (DMF) and 2% N, N- diisopropyl Base ethylamine (DIPEA), room temperature concussion is stayed overnight.High performance liquid chromatography (HPLC) is isolated and purified, mass spectral analysis identification product.NODA The chemical equation for modifying Cx43SP1 is as follows：

NODA modifies Cx43SP1 chemical equation

⁶⁴Cu is marked：Its 10 μ g Cx43SP-NODA is dissolved in the μ l buffer solutions of ammonium acetate 200 of the pH between 4-5, Add 50 μ L's⁶⁴CuCl₂（PH is between 5-6）.Mixed liquor reacts 1 hour in 37 °C.Radiolabeled product is through putting Penetrating property detector-high performance liquid chromatography (RP-HPLC) is isolated and purified, and determines mark rate, radiochemically pure, specific activity.⁶⁴Cu is marked NODA-Cx43SP1 chemical equation is as follows：

⁶⁴Cu marks NODA-Cx43SP1 chemical equation

2）¹⁸F marks Cx43SP

¹⁸F is clinical the most frequently used radionuclide, using compatibility¹⁸F-F^-Direct labeling polypeptide difficulty is very big, typically Using by modification¹⁸F precursors, such as 4- nitrobenzene 2- [¹⁸F] fluorine propionic acid（¹⁸F-NFP）.By this technology, it can obtain being adapted to use It is imaged in heart and tumour Cx43 targeted moleculars¹⁸The characteristics of Cx43 probes of F marks, probe is latent with more clinical practice Power.

¹⁸F-FP-Cx43SP synthesis：

¹⁸The precursor of F marks¹⁸F-NFP (4-nitrophenyl 2- [18F]-fluoropropionate, Rt=22- 23min), the Cx43 targetings μ g of binding peptide 500 are dissolved in 150 μ L anhydrous dimethyl sulphoxides（DMSO）In, and add¹⁸F-NFP and 20 μ L DIPEA（DIPEA）.Mixed liquor reacts the 5% acetic acid neutralization that 800 μ L are added after 30 minutes at room temperature.Product Purified through semi-preparative HPLC C-18 posts.Purified product adds the dilution of 20 μ L water.5mL ethanol and 10mL water are used in preparation process C-18 pillar eluted products are rinsed repeatedly, and final product is dried up at 60 °C with nitrogen.Finally,¹⁸The polypeptide of F marks is dissolved in PBS And by 0.22 μm of hyperfiltration into disinfectant measuring bottle, for external and experiment made on the living.Equally, mark rate is determined with HPLC, Radiochemically pure, specific activity etc..¹⁸F marks Cx43SP1 chemical equation is as follows：

¹⁸F marks Cx43SP1 chemical equation

¹⁸F-AlF-NODA-Cx43SP synthesis：

With NODA-Cx43SP, NODA-Cx43SP is synthesized.Using QMA-SepPak posts absorption 30mCi (1.1GBq)¹⁸F- fluorine Change aluminium, rinsed with the 2.5mL non-metallic ion aqueous solution¹⁸F- aluminum fluorides, and eluted with 400 μ L 0.4M KHCO3 solution¹⁸F- Aluminum fluoride, takes 200 μ L's¹⁸F- aluminun fluoride solutions are standby.Solution ph is adjusted to 4.0 with the acetic acid of not metal ion.Xiang Rong Aluminium chloride is sequentially added into liquid（AlCl₃, 2mM, 3 μ L are dissolved in 0.1M sodium-acetate buffers, pH4) and 5 μ L NOTA- Cx43SP（60mg/mL is dissolved in DMSO).After 100 °C of reactant mixture is incubated 15 minutes, diluted with 1mL non-metallic ions water, production Thing prepares HPLC purifying through half, collects¹⁸F-AlF-NOTA-Cx43SP analogs, are evaporated, and are dissolved in PBS and super by 0.22 μm Microfiltration is into disinfectant measuring bottle, for external and experiment made on the living.Equally, mark rate, radiochemically pure, than work are determined with HPLC Degree etc.,¹⁸F marks Cx43SP1 chemical equation is as follows.

¹⁸F marks Cx43SP1 chemical equation

Except¹⁸, can also be by other outside F-NFP¹⁸F precursors（It is exactly auxiliary group）, marked using similar method Cx43SP.Conventional¹⁸F labelled precursors structural formula [¹⁸F]FBA:[¹⁸F] o-fluorobenzoic acid；[¹⁸F]FSB：[¹⁸F] fluorobenzoate； [¹⁸F]FBEM[¹⁸F] FBBO is as follows：

Except¹⁸Outside F, it can also apply¹¹C、¹³N、¹⁵The radionuclides such as O, choose corresponding precursor, and Cx43SP is carried out Mark.The advantage of the probe of the radioisotope labeling of positron emission：Sensitiveness is high, can accurate quantitative analysis, can clinic convert, spy Pin molecular weight is small, and pharmacokinetic profile is good.

3）：^99mTc marks Cx43SP

Citing：^99mTcO³⁺Or^99mTcO²⁺, bifunctional chelating agent is used as using HYNIC.Comprise the following steps that：

1. the amino on bifunctional chelating agent HYNIC-NHS and Cx43SP（-NH₂）Reaction, using DMF as solvent, 2%DIPEA As catalyst, through high pressure liquid phase (HPLC) after purification, product, mass spectrum is confirmed.

2. technetium-99 m labeled HYNIC-Cx43SP：By the scheme of the two end number mixing ligand complex of " 3+1 ", with tridentate ligand Tricine makees collaboration reagent, with stannous chloride (SnCl₂·H₂O）Make after reducing agent, room temperature reaction 20min, determine mark chemical combination The radiochemical purity of thing, the colloid of casting is less than 1%.

3. analysis：With the method for thin-layer chromatography (ITLC) and high pressure liquid phase (HPLC) come colloid in evaluation of markers compound The content of content and product.

^99mTc marks Cx43SP1 chemical equation is as follows：

^99mTc marks Cx43SP1 chemical equation

According to different^99mTc- cores, select different connectors, by different courses of reaction, can obtain different structure Product.^99mTc marks Cx43SP, common bifunctional chelating agent and the probe of synthesis, and it is as shown in table 2 that R represents Cx43SP：

Table 2

4）^123/124I marks Cx43SP

The appropriate oxidant of selection, connecing can be marked at iodine radioisotope on Cx43SP.Mainly there are 2 kinds of mark sides Method, directly mark and indirect labelling.

1. directly mark：Iodination reaction can be realized directly by " iodine protonation ".^123/124I directly marks Cx43SP normal The oxidant structural formula of use is as follows：

^123/124I directly marks Cx43SP1 chemical equation as follows：

2. indirect labelling：^123/124I indirect labellings Cx43SP frequently with oxidant structural formula it is as follows：

^123/124I indirect labellings Cx43SP1 chemical equation is as follows：

Embodiment 4：The preparation of the Cx43 targeted molecular probes of optical markings

1）Near infrared fluorescent dye marks Cx43SP1

Wavelength 700-900nm near infrared fluorescent dye due to penetration power it is stronger, reach deeper part tissue, therefore It is commonly used to carry out chemiluminescence assay.Conventional near infrared fluorescent dye is shown in figure.Different bifunctional chelating agents may be selected, nearly IR fluorescent dyes are marked on Cx43SP.Cyanine dye chemical structure of general formula is as follows：

The chemical constitution of conventional 4 class near infrared fluorescent dyes is as follows：

(1a) cyanine, (1b) ICG, (1c) SIDAG, (1d) PPCy

Cyanine dye chemical structure of general formula

Cy5.5 is marked：Cx43 specific binding peptides are dissolved in 100 μ L DMSO, with Cy5.5-NHS under the conditions of lucifuge（1 equiv.）Mixing, is codissolved in 2% DIPEA, room temperature, and vibrations are incubated overnight.Product prepares HPLC C18 pillars (250 through half × 10mm) isolate and purify, product is collected, is freezed, yield is calculated, uses mass spectrum（MALDI-TOF-MS）Determine molecular weight.Cy5.5 is marked Remember that Cx43SP1 chemical equation is as follows：

2）, near-infrared nano material mark Cx43SP

The most frequently used at present have a following four classes nano material, including：Include nano-probe, the amount of near infrared fluorescent dye Sub- point, CNT, gold nanoclusters.

By taking near-infrared quantum dots as an example, using quantum dot-labeled Cx43SP：Cx43SP1000eq is taken, it is soluble in water, with 1eq Quantum dot mixing, add 1000eq EDC, be stirred at room temperature reaction 1 hour, with PD10 pillars separate, concentrated with centrifuge tube, survey Concentration.500-600 Cx43SP molecule can be marked on one quantum dot.The probe of synthesis is lived through near-infrared fluorescence imaging Physical examination is surveyed.

Embodiment 5：The preparation of the Cx43 targeted molecular probes of magnetic mark

1）Superparamagnetic nanoparticle（Fe₃O₄）

Cx43SP synthesising probing needles are marked using superparamagnetic nanoparticle, for magnetic resonance imaging.With super-paramagnetism nano Exemplified by particle, Cx43SP is marked using superparamagnetic nanoparticle：Cx43SP 1000eq are taken, it is soluble in water, it is super suitable with 1eq Magnetic nano-particle is mixed, and adds 1000eq EDC, and reaction 1 hour is stirred at room temperature, and is separated with PD10 pillars, dense with centrifuge tube Contracting, surveys concentration.Marked by superparamagnetism iron oxide Cx43SP1 chemical equation is as follows：

2）Paramagnetic metal chelates：, can also be by paramagnetic metal chelates by selecting appropriate bifunctional chelating agent It is marked on Cx43SP, probe is formed, for magnetic resonance imaging.Paramagnetic metal element mainly includes：Gadolinium（Gd³⁺）、Dy³⁺、Tm³ ⁺、Mn²⁺, CEST reagents³He,¹²⁹Xe.Due to paramagnetic metal chelates produce relaxation ability it is weaker, and magnetic resonance molecule into The sensitiveness of picture is relatively low, can also introduce effective signal amplification mechanism, that is, utilizes the macromoleculars, surface such as numerous body, liposome Multiple functional groups are carried, multiple Cx43SP molecules and substantial amounts of paramagnetic metal are chelated into its surface, a probe is formed.It is suitable Magnetic metal chelate labels Cx43SP1 chemical equation is as follows：

Embodiment 6:The preparation of the Cx43 targeted molecular probes of ultrasonic microbubble mark

Cx43SP 1000eq are taken, it is soluble in water, mixed with 1eq ultrasonic microbubble, add 1000eq EDC, be stirred at room temperature Reaction 1 hour.Ultrasonic microbubble mark Cx43SP1 chemical equation is as follows：

Embodiment 7：The preparation of the Cx43 targeted molecular probes of photoacoustic material mark

At present, there are some golden nanometer particles also to can be used to mark Cx43SP, carry out photoacoustic imaging, such as gold nanorods.Should Cx43SP is marked with gold nanorods：Cx43SP 1000eq are taken, it is soluble in water, mixed with 1eq gold nanorods, add 1000eq EDC, be stirred at room temperature reaction 1 hour, with PD10 pillars separate, concentrated with centrifuge tube, survey concentration.Gold nanorods are marked Cx43SP1 chemical equation is as follows：

Embodiment 8：The preparation of the Cx43 targeted molecular probes of multi-mode mark

2 kinds above or a variety of iconography labeling methods are used simultaneously, generation is available for 2 kinds or the inspection of two or more iconography Look into the Cx43 targeting probes of means detection, i.e. multi-mode molecular imaging probe.For example connected in nano-material surface different Functional group, the developer for other patterns such as connecting peptides, radionuclide, fluorescent dyes.

On near-infrared quantum dots surface through sulfydryl and carboxyl modified, upper Cx43SP1 is first connected, quantum is modified with NODA-MAL Point, by the sweet reaction with sulfydryl in Malaysia, by NODA connections over the qds.Use again⁶⁴Cu is marked, radiolabeled product warp PD10 is isolated and purified, and determines mark rate, and radiochemically pure, specific activity, probe is available for near-infrared fluorescence imaging and PET to be imaged simultaneously Detection.⁶⁴Cu and the double mode mark Cx43SP1 of quantum dot chemical equation are as follows：

Embodiment 9：Isolated experiment is proved

1）, Cx43 high-expression cell lines foundation：Cervical Cancer HeLa Cells have been transfected with Cx43 genes, Cx43 is successfully prepared Cervical Tumor cell line HeLa-Cx43 is overexpressed, by for the Hela cells of transgenosis（Without C43 expression）It is used as control.

2）Cy5.5-Cx43SP1 cells are combined and blocking experiment：Hela-Cx43 cells and control group Hela-Control are thin Born of the same parents, 0.5X10⁶/ hole, experiment the previous day is laid on 12 orifice plates, totally four groups：A groups：Hela-Cx43 groups of cells, B groups：Hela- Control groups of cells, C groups：Hela-Cx43 cells blocks groups, D groups：Hela-Control cells blocks groups.Every group of 3 holes, experiment It is repeated 3 times.Cy5.5-Cx43SP1 is added per hole, concentration is 500nmol/L, 1ml.Blocking group adds the Cx43SP1 of no mark, Concentration is 5 μm of ol/L.

As a result show：There is obvious specific binding in Cy5.5-Cx43SP1, with HeLa-Cx43 cells with untransfected The control group HeLa cells of Cx43 genes are then without combination（Fig. 2-a, Fig. 2-b）.Introduce excessive 5 μm of un-marked ol/L's After Cx43SP is blocked, the combination of Cy5.5-Cx43SP and HeLa-Cx43 cells is significantly lowered significantly（Fig. 2-c）.Result above Show, near infrared fluorescent probe Cy5.5-Cx43SP can be over-expressed Cx43 HeLa cell-specifics intake, Cy5.5- Cx43SP and HeLa-Cx43 combination has targeting specific.

Meanwhile, tissue freezing section has further been made, has been imaged under fluorescence microscope.Control is used as using muscle（Fig. 3- a,3-b,3-c）, fluorescence microscopy Microscopic observation probe Cy5.5-Cx43SP is in the distribution situation of heart, tail vein injection Cy5.5- Shown under 1h after Cx43SP, myocardium frozen section fluorescence microscope, Cy5.5-Cx43SP largely assembles in cardiac muscular tissue, and mainly It is distributed in position where the connection of gap between cardiac muscle cell（Fig. 4-a, 4-b, 4-c）.Confirm probe to myocardium gap in tissue level Connection has targeting specific.

3)、⁶⁴Cu-NODA-Cx43SP1 cells are combined and blocking experiment

Hela-Cx43 cells and control group Hela-Control cells, 0.5X10⁶/ hole, experiment the previous day is laid on 12 holes Plate, totally four groups：A groups：Hela-Cx43 groups of cells, B groups：Hela-Control groups of cells, C groups：Hela-Cx43 cells blocks groups, D groups：Hela-Control cells blocks groups.Every group of 3 holes, experiment is repeated 3 times.Added per hole⁶⁴Cu-NODA-Cx43SP1, concentration For 3.2 μ Ci/ holes, 1ml.Blocking group adds the NODA-Cx43SP1 of no mark, and concentration is 50 μ g/ holes（10 times⁶⁴Cu-NODA- Cx43SP1）.As a result show：⁶⁴There is obvious specific binding in Cu-NODA-Cx43SP1, with HeLa-Cx43 cells with not turning The control group HeLa cells of Cx43 genes are contaminated then without combination.Blocking experiment,⁶⁴Cu-NODA-Cx43SP1 and HeLa-Cx43 cells With reference to significantly lowering significantly（Fig. 5）.As a result show ,-NODA-Cx43SP1 can be over-expressed Cx43 HeLa cell-specifics Intake ,-NODA-Cx43SP1 and HeLa-Cx43 combination has targeting specific and high-affinity.

Embodiment 10：, zoopery

1）, probe living biological distribution characteristics：

Bio distributions of the Cy5.5-Cx43SP in normal mouse

Cy5.5-Cx43SP is injected after mono- hour, mouse is put to death, exteriorizes, in vitro major organs near-infrared fluorescent is carried out Imaging display, as a result shows：Probe Cy5.5-Cx43SP is distributed mainly on heart, liver, and intestines and stomach are mainly arranged through liver sausage road Let out, partly through kidney, urinary system excretion.Especially its higher heart intake, this is to express higher Cx43 with normal heart It is very consistent（Fig. 6）.

2）, normal rat PET cardiac imagings：

Previous experiments are in live body level verification⁶⁴Cu-NOTA-Cx43SP normal rat heart targeting ability of aggregation and The biodistribution characteristic of probe.As a result show,⁶⁴Cu-NOTA-Cx43SP 30min after intravenous injection go out in cardia Now significantly build up, be continued until that 3h still has aggregation (Fig. 7), and it is seldom in the aggregation of liver, lungs, and myocardial visualization is clear. Because Cx43 is myocardium major gap connection albumen, as a result illustrate⁶⁴Cu-NOTA-Cx43SP assembles in homing ability to myocardium.Vein is noted Penetrate after 1h, probe exists⁶⁴Cu-NOTA-Cx43SP mouse are distributed mainly on heart, enteron aisle.These results and mouse Cy5.5- The external optical imaging results of Cx43SP are consistent.The experimental result, indicates the Cx43 targeted molecular probes designed by preliminary experiment⁶⁴Cu-NOTA-Cx43S can carry out living imaging to cardiac muscle.

3）, normal mouse PET cardiac imagings：

Carry out simultaneously⁶⁴Cu-NOTA-Cx43SP images experiment in the PET of normal mouse.As a result such as Fig. 8.⁶⁴Cu-NOTA- Cx43SP is now significantly built up after intravenous injection after 1 hour in cardia, while having absorption in liver, and much passes through kidney And bladder drainage.These results have uniformity with rat test, but Heart Imaging result is poor compared with rat.Illustrate probe not In same animal species, bio distribution has different.Meanwhile,⁶⁴Cu-NOTA-Cx43SP probes also have what is further optimized Space and demand.

4）Tumor bearing nude mice optical tumor is imaged：

Xenografts in nude mice model is established using the HeLa-Cx43 cells for being overexpressed Cx43（Fig. 9, left side）, and with Untransfected Cx43 conventional H eLa cell subcutaneous transplantation knurls model is as a control group（Fig. 9, right side）.Carry out live body Cy5.5- Cx43SP1 tumour near-infrared fluorescence imagings, as a result show：Probe Cy5.5-Cx43SP1 is after tail vein injection one hour, in mistake The HeLa-Cx43 tumor locus for expressing Cx43 is assembled in high concentration, and has no and significantly build up in control group HeLa tumor locus.Should As a result show again：In live body level, Cx43 targeted molecular probe Cy5.5-Cx43S have certain to tumour positive Cx43 Targeting specific.

5）, tumor bearing nude mice PET tumor imagings：

Xenografts in nude mice model is established using the HeLa-Cx43 cells for being overexpressed Cx43（Figure 10, left side）.Vein 64Cu-NODA-Cx43SP is injected, concentration is 40 microcuries.Micro PET imaging results show：Probe 64Cu-NODA-Cx43SP After tail vein injection one hour, assemble in the HeLa-Cx43 tumor locus for being overexpressed Cx43 in high concentration.The result shows again Show：In live body level, tumour positive to Cx43 Cx43 targeted molecular probe 64Cu-NODA-Cx43SP has certain target To specificity.

Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims

1. a kind of method of living body molecule imaging, methods described includes：

Cx43 targeting molecule probes are provided, the Cx43 targeting molecules probe targets affine component by signal component, Cx43 And the part of connector three is constituted, the signal group is divided into the part for being available for iconography equipment to detect, the Cx43 targetings are affine Component is the polypeptide portion specifically bound with Cx43, and the connector connects signal component and the affine component of targeting；

With described Cx43 targeting molecules probe to patient position to be measured implement optical imagery, positron emission fault into Picture, single photon emission tomographic imaging, magnetic resonance imaging, photoacoustic imaging, ultrasonic imaging or other live body iconography fusion of imaging skills Art preferred PET/CT or PET/MRI；

The Cx43 targets affine component and Cx43 carboxyl terminal is specifically bound；

The affine component of targeting of the Cx43 is selected from：

Ith, Cx43SP1, Gly-Ala-Pro-Gly-4Hyp-Pro-Tyr

IIth, Cx43SP2, Gly-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly

IIIth, Cx43SP3, its structural formula is as follows：

The connector is selected from DTPA, DOTA, DOTAGA, NOTA, NODAGA, TETA, CB-TE2A, Sar or NODA, or utilizes Other directly chemically react and are directly connected to signal component and the affine components of Cx43；

The signal group of the Cx43 targetings probe is selected from radio isotope, fluorescent dye.

2.Cx43 targeting molecule probe, affine component is targetted and signal component and targeting is close by signal component, Cx43 The part of connector three composition connected with component, the signal group is divided into the part for being available for iconography equipment to detect, described It is the part specifically bound with Cx43 to target affine component, and the connector connects signal component and the affine component of targeting Come；

The affine component of targeting and Cx43 carboxyl terminal are specifically bound；

The affine component of targeting is selected from：

Ith, Cx43SP1, Gly-Ala-Pro-Gly-4Hyp-Pro-Tyr

IIth, Cx43SP2, Gly-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly

IIIth, Cx43SP3, its structural formula is as follows：

The signal group is selected from radio isotope, fluorescent dye；

The connector is selected from DTPA, DOTA, DOTAGA, NOTA, NODAGA, TETA, CB-TE2A, Sar or NODA, or utilizes Other directly chemically react and are directly connected to signal component and the affine components of Cx43.

3. a kind of composition of molecular imaging probe, includes the targeting molecule probe described in claim 2.

4. use of the targeting molecule probe described in claim 2 in the reagent for preparing diagnosis Cx43 abnormal expression relevant diseases On the way.

5. purposes according to claim 4, it is characterised in that the Cx43 abnormal expressions relevant disease is all adjoint Cx43 expresses the congenital or acquired disease day after tomorrow with dysfunction.

6. purposes according to claim 5, it is characterised in that the Cx43 abnormal expressions relevant disease is tumour or painstaking effort Guard system disease is preferably arrhythmia cordis.